The role of estradiol 17 (E2) and estrone (E1) - the two most abundant estrogens in humans - in men's health and disease remains poorly understood. Although both low and high E2 levels have been associated with adverse health outcomes in men, concerns about the accuracy of direct immunoassays for E1 and E2 has clouded interpretation of available data. Even though E1 is as abundant in circulation as E2, little is known about the association of E1 with outcomes in men. Reference limits for total and free E1 and E2 levels are essential for clinical decision making. In the absence of rigorously-derived reference ranges, the partitioning of total and free E1 and E2 levels into normal, low, or high values has been fraught with substantial risk of misclassification. The objective of this collaboration among investigators from Boston University, the Framingham Heart Study (FHS), the European Male Aging Study (EMAS), the Osteoporotic Fractures in Men Study (MrOS), the Concord Health and Ageing in Men Project (CHAMP), the Mayo Clinic, and the Centers for Disease Control is to generate reference limits for total and free E1 and E2 levels in a healthy reference sample of young men in the FHS third generation (Gen 3) cohort. We also will generate age-adjusted reference limits (Z-score approach). Total E1 and E2 levels will be measured using liquid chromatography tandem mass spectrometry (LC-MS/MS) and free E1 and E2 will be calculated. Reference limits generated in FHS Gen 3 will be applied to men in 4 geographically distinct cohorts - FHS Gen 3 plus FHS Offspring cohort (Gen 2) (FHS broad sample), EMAS, MrOS, and CHAMP cohorts. We will relate total and free E1 and E2 levels to diabetes, cardiovascular disease, bone mineral density, physical function, and sexual symptoms, adjusting for age, body mass index, waist circumference, blood pressure, smoking, lipids, glucose, diet and physical activity. We will assess the heritability of E1 and E2 levels to evaluate the contribution of genetic effects on inter-individual variability in E1 and E2 levels. Baseline total and free E1 and E2 as well as longitudinal changes in E2 and E1 will be related to the incidence of adverse outcomes (primary: mortality, diabetes mellitus, cardiovascular disease events~ secondary: progression of functional limitations and disability, osteoporosis, nonvertebral fractures) during longitudinal follow-up. The recommendations for partitioning of men into those with normal, low and high E1 and E2 levels will be guided by considerations of their statistical distribution and the association of outcomes with varying degree of deviations from the reference limits. The proposal will advance our understanding of the role of E1 and E2 levels in men and provide a standardized framework for the interpretation of E1 and E2 levels by practicing physicians. The project is highly cost effective because it leverages existing outcomes data from four well characterized cohorts. The use of LC-MS/MS assay, a standard calibrator, and a community-based reference sample, the availability of 4 geographically distinct validation cohorts, and an inter-disciplinary team of investigators would maximize the chances of success.